My goal in seeking a K08 Career Development Award is to become an independent investigator and leader in the field of liver tissue engineering. I have demonstrated a sustained commitment to become a clinician- scientist, having completed an M.D.-Ph.D. combined degree at Harvard Medical School, General Surgery Residency at the University of California, San Francisco (UCSF), and two years of post-doctoral research investigating three-dimensional (3D) culture as a novel approach to tissue engineering. I completed a clinical fellowship in Minimally Invasive Surgery and have been recruited to UCSF as Assistant Professor of Surgery with 75% protected research time. As a surgeon-scientist, my long-term career goal is to tissue engineer a functional liver unit for therapeutic implantation, preferably by minimally invasive techniques. This award will help me achieve that goal by supporting development of my independent research program based on my post- doctoral work on 3D hepatocyte culture in solid-body rotational bioreactors. Liver transplantation is currently the only treatment for patients with end-stage liver disease (ESLD), which is the 12th leading cause of death by disease in the U.S. The shortage of donor organs remains a major treatment limitation. Alternatives such as hepatocyte transplantation have shown promise in treating metabolic liver disorders, but low engraftment efficiency and poor long-term efficacy are barriers to broader clinical application. Another strategy is ex vivo tissue engineering of a functional livr unit that can be implanted. However, progress thus far towards building 3D tissue structure with biocompatible scaffolds has been hindered because the slow degradation rates of these materials prevent normal cell-cell and cell-to- extracellular matrix (ECM) interactions. My post-doctoral research demonstrated that hepatocytes cultured in Rotating Wall Vessel (RWV) bioreactors, which provide minimal shear stress with maximal 3D spatial freedom, produced self-aggregated spheroids with optimal metabolic and synthetic gene expression and function as compared to those cultured in 2D monolayers. This proposal builds upon those findings and aims to define the role of matrix and stromal cells in maintaining hepatocyte functions and to determine the underlying mechanisms. The research is driven by the hypotheses that 1) 3D cell shape and matrix rigidity are interconnected factors that together modulate hepatocyte function through cytoskeletal tension mediated by the Rho-ROCK and integrin signal cascades, and 2) 3D co-culture of hepatocytes with stellate cells will generate organoids demonstrating optimal scaffold matrix rigidity, ECM composition, and paracrine crosstalk for maintaining hepatocyte functions. Specifically, this proposal will 1) Determine the effect of matrix rigidity on hepatocyt function and the underlying molecular mechanisms and 2) Characterize the matrix rigidity, ECM composition, and paracrine crosstalk of hepatocyte-stellate cell 3D aggregates and the underlying mechanisms required to maintain hepatocyte specific functions. The results of this study are expected to advance the field of tissue engineering by increasing our understanding of hepatocyte cellular response to matrix stiffness and 3D stromal cell interactions. This research will provide the necessary preliminary data for me to initiate an R01-funded study of liver tissue engineering. Importantly, other training activities will be utilized to increase my knowledge in various disciplines important to my career goal of becoming an independent investigator in liver tissue engineering. The research and training will be carried out at UCSF, a top-ranked tertiary referral hospital and internationally-known institution for biomedical research with an incredibly rich environment for scientific investigation and collaboration. I will pursue didactic coursework in extracellular matrix scaffolds and bioengineering through the UCSF Biomedical Sciences Graduate Program. I will attend seminars offered through UCSF's Hepatology Fellowship Program in the Division of Gastroenterology and study liver pathology with a world-renowned liver pathologist. My primary mentor is Dr. Hobart Harris, who is Chief of the Division of General Surgery and an accomplished surgeon-scientist with an extramurally-funded basic science laboratory. Dr. Harris is a well- suited scientific mentor with extensive research background in primary hepatocyte biology and an invaluable overall career mentor in guiding me through the challenges of becoming a surgeon-scientist. The proposed project is interdisciplinary and my co-mentor Dr. Valerie Weaver will provide expertise in extracellular matrix, cytoskeletal, and Rho-ROCK/integrin signaling manipulation. My other co-mentor, Dr. Holger Willenbring, will provide additional expertise in primary hepatocyte and stellate cell culture and functional assays. In addition, I will be working with Dr. Jacquelyn Maher, who is a member of my advisory committee and director of the UCSF Liver Center. Dr. Maher and the UCSF Liver Center will be important resources to support the isolation and analysis of primary hepatocytes and stellate cells proposed in this research. With this mentoring team and UCSF's rich research environment, success of my proposed research and career development are very high. Support through the K08 Career Development Award will be invaluable in helping me achieve my long-term career goal of becoming an independent surgeon-scientist with a research focus in liver tissue engineering. PUBLIC HEALTH RELEVANCE: Liver transplantation is currently the only treatment for patients with end-stage liver disease, but the shortage of donor organs remains a major treatment limitation. In order to develop functional ex vivo engineered liver tissues for organ replacement therapy, this research proposes to investigate the effect of matrix rigidity and three-dimensional stromal cell interactions on hepatocyte function.